PROJECT SUMMARY/ABSTRACT Defects in sperm or oocytes lead to infertility and reproductive failure. For example, chromosomal abnormalities in sperm or oocytes cause of miscarriage and mental retardation, while motility defects in sperm are a common cause of male infertility. Key regulators of fertility, sperm-specific PP1 phosphatases, are required for segregating DNA during spermatogenesis, acquiring motility, and initiating embryo development in species from humans to worms. Because these processes are challenging to study deep within mammalian tissues, we will use the molecular tools available in the transparent model organism C. elegans to define how sperm-specific PP1 phosphatases regulate multiple distinct processes vital for proper sperm formation. PP1 phosphatases are signaling molecules regulated by partners that shift PP1 localization to different subcellular sites to dephosphorylate specific targets that segregate sperm chromosomes, regulate sperm motility, or cue embryo development. However, the partners, targets, and pathways used by PP1s in each of these processes are not well understood. Our first aim is to identify new fertility regulators that function with PP1 in male fertility and embryogenesis. We will use proteomic analysis to identify interacting partners and targets of PP1 phosphatases. Our second aim will characterize the function and localization of a prioritized set of candidate fertility factors in sperm and embryo formation, which are challenging to conduct in other systems. We will prioritize candidates with mammalian homologs and use tools easily implemented in C. elegans, including CRISPR/Cas9 gene editing and the auxin-inducible degradation system, to achieve loss-of-function of candidate factors. We will then use our expertise in sperm development to rapidly catalog functions candidates play in sperm meiosis, motility, and embryogenesis. Cytological analysis and live imaging will determine when and where candidate fertility factors localize in relation to sperm-specific PP1s. These defined localization patterns can serve as diagnostic tools for tracking proper sperm and embryo development. This approach uses well-characterized techniques and reagents available in C. elegans and expert collaborators in a coordinated strategy to discover the larger pathways that sperm-specific PP1 phosphatases use to regulate reproduction across species. We will elucidate molecular pathways for how phosphorylation coordinates distinct fertility processes, a long-standing question in the field. These studies have potential to advance the development of diagnostics and treatment options to avoid the consequences of infertility, chromosomal abnormalities, and reproductive failure.